Use of gpr120 agonists in the prevention or treatment of intestinal-liver immunological damage induced by ethylamino acetate poisoning in poultry

Abstract

The application discloses application of a GPR120 agonist in preventing or treating intestinal-liver immunological injury induced by poultry acetochlor poisoning. According to the intestinal-liver toxicity of chicken acetochlor, the normal expression of G protein-coupled receptor 120 is mainly changed. Subsequently, programmed cell death occurs in the intestinal tract and the liver, and a large amount of inflammatory factor release is caused; the intestinal-liver axis immunological injury is improved by activating the G protein-coupled receptor 120 in vivo and in vitro, so as to rescue the chicken acetochlor poisoning reaction. The application provides a prevention and treatment basis for poultry acetochlor poisoning immunological injury diseases, provides medical data for developing poultry immunological activity drugs or supplements, and also provides more technical means for developing and utilizing environment-friendly, efficient, targeted and slow-release new veterinary drug preparations.

Description

Technical Field

[0001] This invention relates to the application of GPR120 agonists in the preparation of drugs for the prevention or treatment of enterohepatic immune damage induced by acetochlor poisoning in avian animals. This invention belongs to the field of biotechnology. Background Technology

[0002] Acetochlor (ACT) is a broad-spectrum herbicide widely used in agriculture and horticulture for weed control. Its main mechanism of action is to inhibit plant growth and development by interfering with plant cell growth and division, thus achieving weed control. Due to its high efficiency, low toxicity, and broad spectrum, acetochlor is widely used globally. However, the use of acetochlor has also raised concerns about its potential toxicity and impacts on the environment and organisms. Acetochlor can inhibit the growth of certain microorganisms in the soil, affecting soil ecological functions and organic matter decomposition processes. Long-term use may lead to residues in the soil, impacting the stability of soil ecosystems; acetochlor may enter water bodies through runoff and seepage, causing toxicity to aquatic plants and animals and adversely affecting the structure and function of aquatic ecosystems; as a non-selective herbicide, acetochlor may negatively affect non-target plants, and long-term use may lead to a reduction in some plant populations, thus affecting ecosystem plant diversity. More seriously, acetochlor can be transferred through the food chain, impacting livestock and wildlife. Some animals are affected by the toxicity of acetochlor after consuming plants containing it or drinking contaminated water. Furthermore, long-term use of acetochlor may lead to herbicide resistance in some weeds, reducing its effectiveness and increasing the difficulty of crop management.

[0003] The gut-liver axis plays a crucial role in the physiological processes of poultry. Harmful substances exposed to the environment can be absorbed through the intestines. The liver, as the primary detoxification organ, protects the body from toxic damage by metabolizing and eliminating harmful substances. The close connection between the gut and liver ensures adequate digestion and effective absorption of nutrients, maintains electrolyte balance and normal immune system function, and regulates energy metabolism and detoxification. These collaborative functions contribute to maintaining the health and productivity of poultry. Some foods may contain toxic chemicals such as pesticide residues, heavy metals, and plasticizers. These toxins, once ingested, can damage the intestinal mucosa, interfere with nutrient absorption and immune function, and disrupt the balance of gut microbiota. While these toxic chemicals can be broken down and eliminated by the liver's detoxification function, long-term exposure can lead to increased liver burden, disruption of the body's dynamic balance, and complex organ damage or even failure.

[0004] 16S rRNA analysis of the gut microbiota showed that acetochlor poisoning in chickens affected the diversity of the gut microbiota, altering bacterial abundance at the phylum and family levels. BugBase analysis of gut microbiota function revealed a significant increase in Gram-negative bacteria, biofilm formation, and antioxidant stress tolerance after acetochlor exposure. Transcriptomic analysis identified 2233 differentially expressed genes, of which 1223 mRNAs were significantly upregulated and 1010 mRNAs were significantly downregulated. KEGG and GO analysis of differentially expressed genes in the liver transcriptome mainly enriched in immune response regulation signaling pathways, the NF-κB pathway, inflammatory bowel disease, the IgA-producing gut immune network, T-cell receptor signaling pathways, Th17 immune cell differentiation, and even cancer pathways. KEGG enrichment analysis of differentially expressed metabolites showed that the main functional pathways were oxidative phosphorylation, cysteine ​​and methionine metabolism, and glycerophospholipid metabolism. Among them, the G protein-coupled receptor 120 (GPR120), also known as "FFAR4" or "free fatty acid receptor", was significantly underexpressed in the acetochlor treatment group, which attracted our attention.

[0005] GRP120 is a G protein-coupled receptor widely expressed in the gut and liver. It is identified as a fatty acid receptor, particularly for some long-chain fatty acids, such as omega-3 polyunsaturated fatty acids, including EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). Activation of GPR120 can promote insulin sensitivity, regulate lipid metabolism, and improve insulin resistance. GPR120 participates in the regulation of immune cells, especially in the suppression of inflammatory responses. Activation of GPR120 can reduce inflammatory responses and inhibit the production of inflammatory cytokines, thereby combating chronic inflammation-related diseases. With the rapid development of the agricultural and forestry economy, the global use and proportion of herbicides have increased dramatically. Acetochlor, as the most widely used amide herbicide, is the most widely used herbicide in dryland farming in China, especially in Northeast China, with an application area five times larger than that of the triazine herbicide atrazine. Although acetochlor was initially developed for its low toxicity and high efficiency in weed control, its misuse has led to widespread residues in food, water, and the environment. The International Agency for Research on Cancer (IARC) of the World Health Organization classified acetochlor as "probably human carcinogen (Group 2A)" in 2015. Subchronic acetochlor residues may have adverse effects on the body. Current research has focused less on acetochlor poisoning in poultry. Therefore, proper husbandry practices and nutritional support are crucial for maintaining poultry health and normal intestinal and liver function. Summary of the Invention

[0006] The purpose of this invention is to provide a drug that can treat or prevent entero-hepatic immune damage induced by acetochlor poisoning in poultry.

[0007] To achieve the above objectives, the present invention employs the following technical means:

[0008] This invention investigates the mechanism by which acetochlor residues in poultry feed damage the health and production performance of broilers, identifying abnormally expressed factors after acetochlor poisoning and thus revealing the intestinal-hepatic axis toxicity pathway and cell death patterns. Next, by screening beneficial nutrients as antagonistic effectors and activating GPR120 in in vitro LMH and HD11 cell lines, this invention clarifies a feasible approach to rescue acetochlor intestinal-hepatic axis toxicity. Finally, addressing the critical issue of the lack of multifunctional feed-derived toxin detoxifiers in my country, this invention aims to increase GPR120 expression from the dietary source, thereby activating GPR120 and preventing or improving immune damage in chickens.

[0009] Based on the above research, this invention proposes the application of G protein-coupled receptor 120 (GPR120) agonists in the preparation of drugs or feeds for avian acetochlor poisoning-induced entero-hepatic immune damage.

[0010] Preferably, the G protein-coupled receptor 120 agonist is the GPR120 agonist GSK137647A or an omega-3 polyunsaturated fatty acid.

[0011] Among them, the preferred manifestations of entero-hepatic immune damage after acetochlor poisoning in birds are: changes in the structure of the intestinal microbiota and excessive accumulation of inflammatory cells caused by pyroptosis; the liver manifestations are abnormal liver function, low expression of G protein-coupled receptor 120, oxidative phosphorylation, high expression of NF-κB pathway and abnormal expression of immune damage-related pathway proteins.

[0012] Preferably, the G protein-coupled receptor 120 agonist improves bodily damage and immune damage to the intestine and liver by activating GPR120 in vitro and in vivo and blocking programmed cell death.

[0013] Preferably, the G protein-coupled receptor 120 agonist is added to poultry feed to increase the expression of GPR120 from the dietary source, thereby activating GPR120 and preventing or treating entero-hepatic immune damage induced by acetochlor poisoning in poultry.

[0014] Compared with the prior art, the beneficial effects of the present invention are:

[0015] This invention is based on the fact that the enterohepatotoxicity of acetochlor in chickens primarily alters the normal expression of G protein-coupled receptor 120. Subsequently, programmed cell death occurs in both the intestine and liver, triggering the release of a large number of inflammatory factors. By activating G protein-coupled receptor 120 in vitro and in vivo, the invention aims to improve the immune damage of the enterohepatic axis, thereby rescuing the chicken acetochlor poisoning response. This invention provides a basis for the prevention and treatment of immune-damaging diseases caused by acetochlor poisoning in poultry, offers fundamental medical data for the development of immune-active drugs or supplements for poultry, and also provides more technical means for developing and utilizing environmentally friendly, highly efficient, targeted, and sustained-release novel veterinary drug formulations. Attached Figure Description

[0016] Figure 1 Visual lesions observed during intestinal and liver necropsy after exposure to toxins;

[0017] Among them, A: Liver damage manifestations at different doses of acetochlor; B: Intestinal damage manifestations at different doses of acetochlor;

[0018] Figure 2 Morphological changes in intestinal tissue after exposure to the toxin;

[0019] Among them, A: observation of intestinal H&E staining sections; B: observation of intestinal transmission electron microscopy; C: detection of intestinal tight junction marker proteins; D: bar chart of intestinal tight junction marker protein expression levels;

[0020] Figure 3 This refers to the pathological damage to the liver following exposure to toxins.

[0021] Among them, A: observation of liver H&E staining; B: observation of liver Masson staining; C: bar chart of collagen fiber area ratio; DF: serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bile acids (TBA).

[0022] Figure 4 Changes in the gut microbiota structure after exposure to the toxin;

[0023] Among them, A: Bar chart of the gut microbiota with the most significant enrichment; B: Venn diagram of gut species; C: Bar chart of gut community; D: Heat map of gut community;

[0024] Figure 5 The changes in liver transcriptomics after exposure to toxins;

[0025] Among them, A: Liver differential gene volcano plot; B: Liver gene expression density map; C: GO enrichment analysis; D: KEGG enrichment analysis;

[0026] Figure 6 Changes in liver metabolomics after exposure to toxins;

[0027] Wherein, A and B: KEGG metabolic enrichment analysis; C: PCA analysis of expression level distribution; D: pie chart of metabolite proportion;

[0028] Figure 7 The effects of acetochlor on intestinal inflammatory damage;

[0029] The table shows the following data: A: mRNA levels of TLR4, MyD88, p-IκBα, NF-κB, NLRP3, Caspase-1, GSDMD, and ASC; B: heatmap analysis of TLR4, MyD88, p-IκBα, NF-κB, NLRP3, Caspase-1, GSDMD, and ASC; C: protein levels of p-IκBα, NF-κB, NLRP3, Caspase-1, GSDMD, ASC, IL-1β, and IL-18; D: protein quantification bar chart; E: mRNA levels of IL-1β, IL-18, IL-6, IL-8, IL-10, and IL-17; F: heatmap analysis of IL-1β, IL-18, IL-6, IL-8, IL-10, and IL-17.

[0030] Figure 8 The effect of acetochlor (ACT) on the growth of LMH cells in vitro;

[0031] Among them, A: Effect of 12 and 24hACT on LMH cell viability; B: Effect of 48hACT on LMH cell viability; C: Bar graph of average ROS fluorescence intensity; D: Observation of ROS fluorescence staining;

[0032] Figure 9 The effect of acetochlor on the growth of HD11 cells;

[0033] Among them, A: Effect of acetochlor on HD11 cell survival rate at 24h; B: Effect of acetochlor on ROS production in HD11 cells; C: Bar graph of average ROS fluorescence intensity.

[0034] Figure 10 The effects of acetochlor, NAC, and MCC950 on the NF-κB / NLRP3 pathway of LMH;

[0035] Among them, A: GSDMD immunofluorescence staining observation; B: GSDMD average fluorescence intensity; C: NF-κB / NLRP3 pathway protein expression detection;

[0036] Figure 11 The effects of acetochlor on liver ultrastructure and mitochondrial activity;

[0037] Among them, A: Transmission electron microscopy observation of liver; B: Staining observation of LMH cell mitochondrial activity probes; C: Bar graph of average fluorescence intensity of mitochondrial activity.

[0038] Figure 12 The effects of acetochlor, NAC, MCC950, and GSK-7A on cell damage;

[0039] Among them, A: Mitochondrial membrane potential staining observation; B: Mean fluorescence intensity of mitochondrial membrane potential; C: Flow cytometry detection; D: Bar chart of cell death percentage;

[0040] Figure 13 The effects of acetochlor, NAC, MCC950, and GSK-7A on the expression of surface markers in M1-HD11 cells;

[0041] Among them, A: CD86 immunofluorescence staining observation; B: CD86 average fluorescence intensity bar graph; C: M1 marker factor IL-1β and TNFα mRNA bar graph;

[0042] Figure 14 The effects of acetochlor, NAC, MCC950, and GSK-7A on the expression of surface markers in M2-HD11 cells;

[0043] Among them, A: CD163 immunofluorescence staining observation; B: CD163 average fluorescence intensity bar graph; C: M2 marker factor IL-10 and TGFβ mRNA bar graph;

[0044] Figure 15 The protective effect of dietary Omega-3 against acetochlor-induced intestinal damage;

[0045] Among them, A: observation of intestinal HE staining; B: observation of intestinal transmission electron microscopy; C: detection of intestinal tight junction marker proteins; D: bar chart of tight junction protein expression.

[0046] Figure 16 The protective effect of dietary Omega-3 against acetochlor-induced liver injury;

[0047] Among them, A: observation of liver HE staining; B: observation of liver collagen fiber staining; C: bar chart of collagen fiber proportion; D: observation of liver transmission electron microscopy. Detailed Implementation

[0048] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0049] Example 1: Establishment of a chicken acetochlor poisoning model

[0050] The in vivo replication of the toxicity model and the establishment of a gradient toxicant attack dosage model are detailed below:

[0051] Forty one-day-old broiler chickens were obtained from the laboratory animal center and subjected to a one-week acclimatization period. For the first seven days, the temperature was 33°C, humidity was 65%, and light was approximately 16 hours per day. Thereafter, the temperature was decreased by 1°C every three days, and the humidity was decreased by 5% weekly, maintaining a humidity level of 55%. They were then randomly assigned to four groups: a control group (Con, basal diet without ACT), a low-dose group (LACT, 5 mg / kg ACT, which is 0.5 times the maximum residue limits (MRLs) for chicken feed (dry matter) as specified by the Joint Conference on Pesticide Residues (JMPR) and the National Food Safety Standard – Maximum Residue Limits for Pesticides in Food (GB2763-2021), a medium-dose group (MACT, 50 mg / kg ACT), and a high-dose group (HACT, 500 mg / kg ACT). These doses were selected based on 1 / 250 and 1 / 25 of the acute reference dose of ACT estimated by the European Food Safety Authority (EFSA), respectively. After 35 days of ACT treatment, the broiler chickens were euthanized, and their intestinal tissue was collected. The collected samples were fixed in 4% paraformaldehyde solution and 2.5% pentylene glycol solution. The remaining samples were appropriately labeled and stored at ultra-low temperature.

[0052] Example 2: Histological observation and detection of a chicken acetochlor poisoning model

[0053] method:

[0054] 1. The specific pathological damage to the intestinal and liver tissues was observed through necropsy and hematoxylin and eosin (H&E) staining. The steps are as follows:

[0055] The animal was euthanized, and a necropsy was performed along its abdomen. The animal was observed and photographed without damaging its tissue structure.

[0056] The slices were sequentially immersed in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then washed with distilled water.

[0057] Hematoxylin staining of cell nuclei: Slices are stained with Harris hematoxylin for 3-8 min, washed with distilled water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water.

[0058] Stain the cytoplasm with eosin; immerse the sections in eosin staining solution for 1-3 minutes.

[0059] The slices were sequentially immersed in 95% ethanol I for 5 min, 95% ethanol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min to dehydrate and clear the slices.

[0060] Remove the sections from the xylene and let them dry slightly before mounting them with neutral resin.

[0061] Microscopic examination, image acquisition and analysis.

[0062] 2. Observe the ultrastructural changes of tissues using transmission electron microscopy. The specific implementation steps are as follows:

[0063] (1) Pentylene glycol-fixed tissue samples;

[0064] (2) The slicing process is approximately 70 nm;

[0065] (3) The slices were placed in the transmission electron microscope grid;

[0066] (4) The sample was dehydrated using acetone;

[0067] (5) Transmission electron microscopy observation and image acquisition.

[0068] 3. Masson staining

[0069] First, the paraffin sections are dewaxed to remove the paraffin wax. Then, counterstaining is performed, with the nuclei stained with hematoxylin. Next, differentiation occurs to bring the nuclei to a blue color, followed by rinsing to remove excess dye. The crucial staining step is staining with Masson's compound dye solution for 1-2 minutes to bring the collagen fibers to a blue color. Finally, dehydration, clearing, and mounting are performed to complete the staining process.

[0070] 4. Detection of intestinal tight junction proteins

[0071] (1) Sample preparation: Total protein was extracted from intestinal tissue, the tissue was lysed with RIPA lysis buffer (strong), and the concentration was determined using a BCA protein concentration assay kit.

[0072] (2) Protein electrophoresis: Protein samples were separated using SDS-PAGE gel electrophoresis.

[0073] (3) Transfer: Transfer was performed in a Tris-glycine buffer solution containing 20% ​​methanol to transfer the protein blot to a membrane containing nitrate and cellulose to transfer the separated protein from the gel to a PVDF membrane.

[0074] (4) Blocking: Use 5% skim milk powder to reduce non-specific binding, and block at 37°C for two hours before PBST rinsing.

[0075] (5) Primary antibody incubation: The membrane was incubated with the specific primary antibody listed in Table 1 to bind to the target protein. β-actin was used as an internal control protein.

[0076] (6) Secondary antibody incubation: After washing, add horseradish peroxidase-labeled secondary antibody for incubation (see Table 1).

[0077] (7) ECL luminescence: After adding ECL developer, the target protein will appear as a band after exposure imaging.

[0078] (8) Data analysis: Analyze the relative expression level of the target protein.

[0079] Table 1 Information on tight junction protein antibodies

[0080] Antibody Dilution ratio company ZO-1 1:300 Wanleibio Claudin-1 1:500 Wanleibio Occludin 1:500 Wanleibio β-actin 1:1000 Abclonal Goat Anti-Rabbit IgG Antibody 1:10000 Biosharp

[0081] 5. Liver function test

[0082] Add 9 volumes of physiological saline at a tissue weight (g) to volume (mL) ratio of 1:9, homogenize mechanically under ice-water bath conditions, centrifuge at 2500 rpm for 10 minutes, and collect the supernatant for analysis. Add ALT, AST, and TBA indicators respectively, and measure the absorbance of the microplate in a microplate reader.

[0083] result:

[0084] The results are as follows Figure 1-3 As shown, the results indicate that acetochlor exposure leads to progressive tissue damage, including intestinal bleeding, increased inflammatory cells, broken or missing intestinal villi, reduced goblet cells and even extravasation of intestinal contents, loss of tight junctions, and decreased tight junction protein markers. The liver shows focal necrosis, significant hepatocyte swelling and increased collagen fibers, and serum tests show elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bile acids (TBA).

[0085] Example 3: Verification of the effect of acetochlor on 16S rRNA in chicken intestines

[0086] method:

[0087] 1) Collect intestinal samples from chicken acetochlor poisoning model and homogenize the samples. Disrupt cells, use EDTA to lyse them, collect the precipitate after cell lysis, precipitate DNA with 70% ethanol, wash the precipitate with 70% ethanol, centrifuge the precipitate, resuspend the DNA with TE buffer, and then fix one end of the DNA fragment with primer base complementation on the chip.

[0088] 2) Using DNA fragments as templates and the base sequences fixed on the chip as primers, PCR synthesis is performed to synthesize the target DNA fragment on the chip;

[0089] 3) After denaturation and annealing, the other end of the DNA fragment on the chip randomly complements another nearby primer and is also fixed, forming a "bridge".

[0090] 4) PCR amplification produces DNA clusters;

[0091] 5) DNA amplicon linearizes into a single strand.

[0092] 6) Add modified DNA polymerase and dNTPs with 4 fluorescent labels, and synthesize only one base per cycle;

[0093] 7) Use a laser to scan the surface of the reaction plate and read the types of nucleotides that were polymerized in the first round of reaction for each template sequence;

[0094] 8) Chemically cleave the "fluorescent group" and "terminator group" to restore the 3' end stickiness and continue polymerizing the second nucleotide;

[0095] 9) Analyze the fluorescence signal results collected in each round to obtain the sequence of the template DNA fragment.

[0096] Illumina sequencing PE reads are first assembled based on overlap relationships, while sequence quality is controlled and filtered. After sample differentiation, OTU clustering and taxonomic analysis are performed. Based on OTUs, various diversity indices can be analyzed, and sequencing depth can be detected. Based on taxonomic information, statistical analysis of community structure can be performed at various taxonomic levels. Building upon these analyses, a series of in-depth statistical and visualization analyses, such as multivariate analysis and significance testing, can be conducted on the community composition and phylogenetic information of multiple samples.

[0097] result:

[0098] The results are as follows Figure 4 As shown, data analysis in this invention revealed that acetochlor can alter intestinal flora homeostasis. Significantly different bacterial groups were mainly enriched in Firmicutes, Verrucomicrobia, and Desulfobacterota, manifested as a decrease in the core intestinal bacteria genera *Blautia* and *Subdoligranulum*, an increase in *Ruminiclostridium*, an increase in *unclassified f. lachnospiraceae*, an increase in *Romboutsia*, and a decrease in *Akkermansia*.

[0099] Example 4: Verification of the effects of acetochlor on the chicken liver transcriptome

[0100] method:

[0101] (1) Total RNA was extracted from tissue samples of a chicken acetochlor poisoning model. The concentration and purity of the extracted RNA were detected using Nanodrop 2000, RNA integrity was detected by agarose gel electrophoresis, and RIN value was determined by Agilent 2100. For a single library preparation, the total RNA amount was required to be ≥1 μg, the concentration ≥35 ng / μL, OD260 / 280 ≥1.8, and OD260 / 230 ≥1.0.

[0102] (2) Eukaryotic mRNA has a polyA tail at the 3' end. By using magnetic beads with Oligo(dT) to pair polyA with AT bases, mRNA can be isolated from total RNA for transcriptome analysis.

[0103] (3) The Illumina Novaseq 6000 platform is designed for sequencing short sequence fragments. The enriched mRNA is a complete RNA sequence with an average length of several kb, so it needs to be randomly fragmented. Adding a fragmentation buffer can randomly break the mRNA, and small fragments of about 300 bp can be separated by magnetic beads.

[0104] (4) Under the action of reverse transcriptase, six-base random primers are added to synthesize one-stranded cDNA using mRNA as a template, followed by two-strand synthesis to form a stable double-stranded structure.

[0105] (5) The double-stranded cDNA structure has sticky ends. Add EndRepair Mix to make it into blunt ends, and then add an "A" base to the 3' end to connect the Y-shaped adapter.

[0106] result:

[0107] The results are as follows Figure 5As shown, the results indicate that transcriptomics revealed 19,051 differentially expressed genes, of which 1,223 were upregulated and 17,828 were downregulated. GO analysis mainly enriched immune-related pathways such as immune response signaling pathways, antigen receptor-mediated signaling pathways, cell chemotaxis, immune response-activation of cell surface receptors, immune response activation signal transduction, leukocyte proliferation, and leukocyte activation participating in immune responses. KEGG analysis enriched T cell receptor signaling pathways, cell adhesion molecules, chemokine signaling pathways, Th17 cell differentiation, inflammatory bowel disease, NF-κB signaling pathway, intestinal immune network of IgA production, B cell receptor signaling pathway, and NK cell-mediated cytotoxicity.

[0108] Example 5: Validation of the effects of acetochlor on the untargeted metabolome of chicken liver by LC-MS

[0109] method:

[0110] Liver tissue was collected from a chicken acetochlor poisoning model. The tissue was pretreated by grinding, and extraction was accelerated with acetonitrile. After shaking and centrifugation, the supernatant was filtered through a 0.2 μm filter to concentrate the metabolites. Finally, a mixed sample was extracted from all samples and separated by liquid chromatography. Individual components were ionized using a high-vacuum mass spectrometer. The mass-to-charge ratio (m / z) was used to obtain the mass spectra. The mass spectrometry data were then analyzed to obtain qualitative and quantitative results. Specifically, 50 mg of sample was added to 400 μl of extraction solution (methanol:water = 4:1 (v:v)) containing 0.02 mg / mL internal standard (L-2-chlorophenylalanine), followed by freeze-grinding. Low-temperature ultrasonic extraction was performed for 30 min (5℃, 40 kHz). The sample was then centrifuged at -20℃ for 30 min, and the supernatant was analyzed on a computer. The LC-MS analysis was performed using an AB SCIEX ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry (UHPLC-Triple TOF) system.

[0111] result:

[0112] The results are as follows Figure 6 As shown, KEGG metabolomics analysis mainly enriched in the PPAR signaling pathway, ferroptosis, oxidative phosphorylation, TCA cycle, glycerophospholipid metabolism, and linoleic acid metabolism; PCA analysis showed good dispersibility; HMDB analysis of differential metabolites showed that lipids and lipid-like molecules accounted for the highest proportion in the acetochlor group, followed by organic acids and their derivatives, organic heterocyclic compounds, organic oxygen compounds, and benzene compounds, with proportions of 26.95%, 21.28%, 13.48%, 12.06%, and 11.35%, respectively.

[0113] Example 6: Real-time quantitative PCR and Western Blot detection of intestinal inflammatory markers

[0114] method:

[0115] RNA was extracted from chicken acetochlor poisoning model tissues using TRIzol reagent and its instructions in a clean bench. 1 μL of RNA extract was diluted 100-fold with RNAase water, and the RNA concentration was determined by UV spectrophotometry. cDNA synthesis was performed using Taraka; the reverse transcription system and reaction conditions are shown in Table 2 below.

[0116] Table 2 Reverse Transcription System

[0117]

[0118] qPCR validation was performed on the cDNA samples. First, the gene sequence was found in NCBI, and primers were designed using Primer 5.0, with F for Forward and R for Reverse. The primers used for q-PCR detection are listed in Table 3 below.

[0119] Table 3 Primers for qRT-PCR detection

[0120]

[0121]

[0122] Then, use qPCR was performed using a 480 instrument. The system and reaction conditions are shown in Table 4 below:

[0123] Table 4 qPCR reaction system

[0124]

[0125] Last 2 -△△CT The relative content of the target gene is calculated using this method.

[0126] The specific method for detecting inflammation-related marker proteins using Western blotting is the same as in Example 2; antibody information is shown in Table 5.

[0127] Table 5. Information on relevant antibodies

[0128]

[0129]

[0130] result:

[0131] The results are as follows Figure 7As shown, the results indicate that exposure to ACT activates the TLR4 / NF-κB / NLRP3 pathway, enhances the NLRP3 inflammasome and Caspase-1, and further cleaves GSDMD. Subsequently, inflammatory markers such as IL-1β, IL-18, IL-6, IL-8, and IL-17 are released, exacerbating the enteritis response.

[0132] Example 6: Establishment of acetochlor toxicity models in HD11 and LMH cell lines

[0133] method:

[0134] 1. Resuscitate HD11 and LMH cells, mix the cells with complete culture medium by pipetting, and transfer them to culture flasks. Incubate in an incubator at 37°C and 5% CO2.

[0135] 2. After the cells stabilized, they were seeded into 96-well plates for CCK8 assays. Different concentrations of acetochlor standard solution were exposed to HD11 and LMH, and the concentration showing a 25% biological effect (IC50) was measured. 25 ).

[0136] result:

[0137] The results are as follows Figure 8 A and Figure 9 As shown in Figure A, the results clearly demonstrate that exposure to acetochlor triggered a significant inhibition of cell activity.

[0138] Example 7: Detection of ROS accumulation levels in LMH and HD11 cells and validation of the roles of NAC and MCC950 in a cell poisoning model.

[0139] method:

[0140] 1. After exposing LMH and HD11 cells to acetochlor standard solution for 6-24 h, the culture medium was removed, the cells were washed three times with PBS, DCFH-DA (10 μM) was added, and the cells were incubated at 37 °C for 30 min. The cells were then washed twice with PBS to remove the dye, and images were observed and captured using a fluorescence microscope.

[0141] 2. Use ImageJ software to calculate the average fluorescence intensity of cells.

[0142] result:

[0143] The results are as follows Figure 8 C and D Figure 9 As shown in B and C, the results indicate that acetochlor treatment can induce excessive ROS accumulation in both cell types, and the addition of NAC and MCC950 can significantly alleviate this abnormality.

[0144] Example 8: Detection of GSDMD expression level in LMH cells and verification of the roles of NAC and MCC950 in a cell poisoning model.

[0145] method:

[0146] 1. Immunofluorescence labeling method for detecting GSDMD expression level in LMH cells. Cells were seeded in 12-well plates and stained with immunofluorescence for 24 or 48 hours after adhesion. Cells were washed 2-3 times with pre-cooled PBS. Immunofluorescence staining fixative was added for 10 min, followed by 2-3 washes with PBS. Cells were permeated with 0.2% Triton X-100 for 10 min, followed by 2-3 washes with PBS. Cells were blocked with 5% BSA for 1 h. Cells were incubated with GPR120 antibody for 12 h, followed by 1-2 washes with PBS. Cells were incubated with FITC-labeled secondary antibody for 2 h, followed by 1-2 washes with PBS. Cell nuclei were labeled with DAPI for 10 min, followed by 2 washes with PBS. Cells were observed and images were acquired under a fluorescence microscope.

[0147] 2. Calculate fluorescence intensity as in Example 7.

[0148] Similar to Example 7, 1 mM N-acetylcysteine ​​(NAC) and 1 μM MCC950 were added respectively, and GSDMD expression was detected by immunofluorescence and the fluorescence intensity was calculated.

[0149] 3. The method is the same as in Example 2, and the antibody information is the same as shown in Table 5.

[0150] result:

[0151] The results are as follows Figure 10 As shown, this result indicates that acetochlor exposure leads to the accumulation of large amounts of GSDMD fluorescence and promotes the expression of NLRP3, Caspase-1, ASC, and GSDMD proteins. Treatment with NAC and MCC950 can significantly improve this condition, with MCC950 showing better efficacy than NAC. This suggests that inhibition of the NLRP3 inflammasome is key to improving pyroptosis.

[0152] Example 9: Effects of NAC, MCC950, and GSK137647A (GSK-7A for short) on acetochlor mitochondrial membrane potential, mitochondrial activity, and cell death.

[0153] method:

[0154] 1. Mitochondrial membrane potential detection:

[0155] Cells were seeded in 12-well plates at a density of 1×10⁻⁶. 4Cells / mL. Incubate overnight at 37°C in a 5% CO2 incubator. Wash cells once with PBS solution and add 1 ml of cell culture medium. The cell culture medium may contain serum and phenol red. Add 1 ml of JC-1 staining working solution and mix thoroughly. Incubate at 37°C for 20 minutes. After incubation, aspirate the supernatant and wash twice with JC-1 staining buffer. Add 2 ml of cell culture medium, which may contain serum and phenol red. Observe under a fluorescence microscope.

[0156] 2. Mitochondrial activity detection

[0157] Add 1 mM Mito-Tracker Green stock solution to cell culture medium or a suitable solution at a ratio of 1:5000 to achieve a final concentration of 20 nM. The Mito-Tracker Green working solution needs to be pre-incubated at 37°C before use. Remove the cell culture medium, add the prepared Mito-Tracker Green staining working solution (pre-incubated at 37°C) from step 2, and incubate with the cells at 37°C for 45 minutes. Remove the Mito-Tracker Green staining working solution and add fresh cell culture medium (pre-incubated at 37°C). Observe under a fluorescence microscope.

[0158] 3. Flow cytometry:

[0159] First, wash the cells twice with PBS, discard the PBS, add 0.25% trypsin to digest the cells, add complete culture medium to stop the digestion, and collect them in EP tubes; then, centrifuge at 1500 rpm / min for 5 min, wash twice more with PBS, add 250 μL B inding buffer, 5 μL Annexin V-FITC, and 10 μL Propidium, and incubate at room temperature for 20 min; then use a Beckman flow cytometer to detect cell death.

[0160] result:

[0161] The results are as follows Figure 11 and Figure 12 As shown, the results indicate that acetochlor can reduce mitochondrial activity and mitochondrial membrane potential and cause 26.9% cell death. Treatment with NAC, MCC950, and GSK-7A can significantly improve these effects, with GSK-7A showing the best mitigating effect.

[0162] Example 10: Verification of the effect of GPR120 agonist in acetochlor-induced LMH and HD11 poisoning models

[0163] method:

[0164] 1. Treat the two cell lines with 25 μM and 50 μM of GPR120 agonist GSK-7A and acetochlor standard solution for 24 h respectively;

[0165] 2. In the same manner as in Examples 8, 9, and 12, the cell damage, GPR120, and ROS expression were detected sequentially, and the rescue effects of the three therapeutic agents were compared.

[0166] 3. Immunofluorescence assay was used to detect macrophage markers CD86 and CD163. The procedure was the same as in Example 8. Antibody information is shown in Table 5 below:

[0167] 4. qRT-PCR was used to detect TGFβ, IL-10, IL-1β, and TNF-α. Primers are shown in Table 3 and Table 6 below.

[0168] Table 5 Immunofluorescent Antibodies

[0169] Antibody Dilution ratio company CD86 1:200 Abclonal CD163 1:200 Abclonal FITC-IgG(H+L) 1:200 Beyotime

[0170] Table 6 Primers for qRT-PCR detection

[0171]

[0172] result:

[0173] The results are as follows Figure 9 , Figure 12 , Figure 13 , Figure 14 As shown, these results indicate that all three therapeutic agents can improve acetochlor-induced cell damage, ROS accumulation, and pyroptosis. Regarding therapeutic efficacy, NAC was significantly effective only in inhibiting ROS, while MCC950 showed good efficacy only in alleviating pyroptosis. However, activation of GPR120 demonstrated a strong therapeutic advantage, effectively improving both ROS outbreaks and pyroptosis, as well as the abnormal expression of immune factors and macrophage M1 and M2 surface markers.

[0174] Example 11: Validation of a dietary Omega-3 polyunsaturated fatty acid detoxification model

[0175] method:

[0176] 1. A broiler detoxification model was established by adding 5% Omega-3 polyunsaturated fatty acids to the feed source, and the detoxification effect was evaluated.

[0177] 2. Histomorphological observation of the recovery of pathological damage

[0178] The specific steps are the same as in Example 2.

[0179] 3. Western blotting method for detecting tight junction protein expression levels

[0180] Antibodies are the same as Table 1.

[0181] result:

[0182] The results are as follows Figure 15 , Figure 16 As shown, this result indicates that Omega-3 has a strong tissue repair capacity. The pathological damage to tissues caused by acetochlor was significantly restored in the Omega-3 treatment group, mainly manifested as better tight junctions and more regular intestinal villus arrangement, while also restoring inflammatory infiltration. Hepatocellular swelling was well improved in this treatment group, and Omega-3 restored liver collagen fiber deposition.

[0183] In summary, this invention proposes that the poisoning mechanism of the feed-derived toxin acetochlor in poultry mainly disrupts the stability of the intestinal-hepatic axis, inducing immune damage in both. Feed-derived Omega-3 supplementation activates GPR120 expression in chickens after acetochlor poisoning, effectively improving organ damage induced by this toxin. Therefore, antagonistic effectors against immune-damaging diseases induced by the feed-derived toxin acetochlor are the focus of this invention.

Claims

1. The application of G protein-coupled receptor 120 (GPR120) agonists in the preparation of drugs or feeds for the prevention or treatment of entero-hepatic immune damage induced by acetochlor poisoning in poultry, wherein the G protein-coupled receptor 120 agonist is GPR120 agonist GSK137647A or omega-3 polyunsaturated fatty acid.

2. The application as described in claim 1, characterized in that, The entero-hepatic immune damage following acetochlor poisoning in avian animals is manifested as: changes in the gut microbiota structure and excessive accumulation of inflammatory cells due to pyroptosis; the liver is characterized by abnormal liver function, low expression of G protein-coupled receptor 120, oxidative phosphorylation, high expression of NF-κB pathway and abnormal expression of proteins in immune damage-related pathways.

3. The application as described in claim 1, characterized in that, The G protein-coupled receptor 120 agonist described herein improves bodily damage and immune damage to the intestine and liver by activating GPR120 in vitro and in vivo and blocking programmed cell death.

4. The application as described in claim 1, characterized in that, The G protein-coupled receptor 120 agonist is added to poultry feed.

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